Title: Membrane interaction of Mycobacterium tuberculosis virulence factors PROJECT SUMMARY It is estimated that Mycobacterium tuberculosis (Mtb), a contagious and airborne bacterial pathogen, infects one-third of the world population and causes 1-2 million deaths each year. It is believed that initial Mtb infection occurs in alveolar macrophages. Mtb is internalized into the phagosome, where it penetrates the phagosomal membrane and translocates into the cytosol for replicating and cell-to-cell spreading. The cytosolic translocation is regarded as an important mechanism of Mtb pathogenesis. Two Mtb virulence factors, namely 6-kDal early antigenic target (MtbESAT6) and 10-kDal culture filtrate protein (MtbCFP10), are secreted out of Mtb as a heterodimer and have been implicated to play an essential role in Mtb cytosolic translocation. Genetic knockout of either esat-6 or cfp-10 results in defective cytosolic translocation and attenuated virulence. Our recent studies have suggested that MtbESAT-6 has an acidic-pH dependent pore- forming activity that is required for Mtb cytosolic translocation and virulence. However, the molecular mechanisms governing MtbESAT-6 pore formation and heterodimer dissociation are not clear. In the present proposal, using a variety of biochemical, microscopic, structural and cellular approaches, we will probe the dynamic process of MtbESAT-6 pore formation and determine the structure of the pore complex. We will also investigate the role of N?-acetylation of MtbESAT-6 in acidification-induced heterodimer dissociation, a prerequisite for MtbESAT-6 to interact with the membrane. Studies designed in this grant proposal are aimed to fill the critical gap in our understanding of the MtbESAT-6-dependent molecular events during Mtb infection in alveolar macrophages and other phagocytes. Knowledge obtained from the proposed studies will facilitate the development of novel countermeasures, therapeutics and vaccines, against tuberculosis. !